A method of spatial high resolution imaging a structure of a sample, the structure comprising a luminescent marker, is known as STED (Stimulated Emission Depletion) scanning luminescence light microscopy. Here, the sample is at first subjected to focused excitation light exciting the luminescent marker out of an excitable electronic ground state into a luminescent electronic state. Then, the sample is subjected to an intensity distribution of emission stimulation light stimulating the excited luminescent marker for emission of light at the wavelength of the emission stimulation light, i.e. at another wavelength as that one of the luminescence light, and thus de-exciting the excited luminescent marker back into its ground state. The intensity distribution of the emission stimulation light has a local intensity minimum. If the emission stimulation light has de-excited the excited luminescent marker by stimulated emission everywhere outside this local intensity minimum, luminescence light afterwards emitted out of the area of the intensity distribution of the emission stimulation light may only come out of the intensity minimum. Thus, this luminescence light may be assigned to the position of the intensity minimum within the sample. If the minimum is a null or zero point of an interference pattern of the emission stimulation light, for example, increasing the intensity of the emission stimulation light decreases the dimensions of the intensity minimum within which the emission stimulation light does not completely de-excite the excited luminescent marker, i.e. not up a saturation of this de-excitation. By increasing the intensity of the emission stimulation light, the dimensions of the intensity minimum may particularly be made smaller than Abbe's diffraction limit at the wavelength of the excitation light and the luminescence light which delimits the spatial resolution in exciting the sample with the focused excitation light and in imaging the structure of the sample emitting the luminescence light onto an image sensor.
A further method of spatial high resolution imaging a structure of a sample, the structure comprising a luminescent marker, is known as GSD (Ground State Depletion) scanning luminescence light microscopy. In this known method, the sample, prior to being subjected to focused excitation light, is subjected to an intensity distribution of luminescence inhibition light comprising a local intensity minimum, the luminescence inhibition light transferring the luminescent marker into a long-living electronic dark state, like for example a triplet state, out of which it is not excited by the excitation light into a luminescent electronic state. Everywhere outside the intensity minimum of the intensity distribution of the luminescence inhibition light this transfer into the dark state is driven up to saturation. I.e. only in the intensity minimum of the intensity distribution of the luminescence inhibition light, the luminescent marker, after application of the luminescence inhibition light, is still in its electronic ground state out of which it is excited by the excitation light into the luminescent state. Luminescence light emitted by the luminescent marker after the excitation with the excitation light thus comes out of the intensity minimum of the intensity distribution of the luminescence inhibition light and may thus be assigned to the position of the intensity minimum within the sample independently on the spatial resolution in exciting the sample and in imaging the sample onto the detector used.
Both STED and GSD scanning luminescence light microscopy belong to RESOLFT (Reversible Saturable Optical Fluorescence Transitions) scanning luminescence light microscopy. A further method belonging to RESOLFT scanning luminescence light microscopy makes use of a so-called switchable luminescent marker for spatial high resolution imaging of a structure of a sample comprising the luminescent marker. By means of luminescence inhibition light, the switchable luminescent marker is switched out of a first conformation state in which it is effective as a luminescent marker into a second conformation state in which it is not effective as a luminescent marker. Thus, the switchable luminescent marker, in its second conformation state, at least by means of excitation light which is suitable for exciting the switchable luminescent marker its first conformation state, is not excitable into a luminescent electronic state out of which it emits the luminescence light being registered. With a sufficient long lifetime of the second conformation state, only comparatively low light intensities are required to drive this switching up to saturation everywhere outside a local intensity minimum of an intensity distribution of the luminescence inhibition light. Further, there is no significant danger that the luminescent marker transferred into its second conformation state is bleached, as it does not respond to the luminescence inhibition light and the excitation light in its second conformation state.
In all variants of RESOLFT scanning luminescence light microscopy, only luminescence light is registered, which comes out of an area of the sample corresponding to the position of the local intensity minimum of the luminescence inhibition light in the sample. For imaging a larger area of the sample, the sample is scanned with the local intensity minimum of the luminescence inhibition light. From the luminescence light registered during scanning, an image of the larger area of the sample can be generated.
P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell: “Parallelized STED fluorescence nanoscopy”, Opt. Express 19, 23716-23726 (2011) describe an STED scanning fluorescence light microscope in which an intensity distribution of emission stimulation light is composed of four partial intensity distributions arranged side by side. Each of the four partial intensity distributions comprises a local intensity minimum delimited in two dimensions. Further, each of the four partial intensity distributions of the emission stimulation light is superimposed with a partial intensity distribution of excitation light which comprises an intensity maximum at the location of the intensity minimum of the emission stimulation light. The partial intensity distributions are generated in that beams of the emission stimulation light and of the excitation light which are aligned on a common optical axis pass through two Wollaston prisms arranged one behind the other. The Wollaston prisms divide the beams into partial beams which, in pairs of one partial beam of stimulation light and one partial beam of excitation light, propagate in four slightly different directions. These four pairs of partial beams pass through a segmented chromatic phase plate which selectively deforms the wavefronts of the partial beams of the luminescence inhibition light so that these partial beams, when all partial beams are afterwards focused into the sample, form the partial intensity distributions with the intensity minima. Due to the four different directions of the partial beams, the partial intensity distributions are arranged at distances within the sample and define four similar local intensity minima. The luminescence light emitted out of the four local intensity minima is spatially separated and registered. By simultaneously scanning the sample with the four intensity minima of the luminescence inhibition light arranged at distances, the time required for imaging the structure of interest is reduced to a quarter as compared to imaging the structure only using one intensity minimum.
WO 2006/127692 A2 discloses a method of high resolution imaging a structure of a sample, the structure comprising a phototransformable optical label (PTOL), which is also based on the RESOLFT concept. By means of a pulse of activation light, the PTOL is transferred out of an inactive state into an active state. In the active state (in contrast to the inactive state) the PTOL may be excited by excitation light for the emission of luminescence light. The sample is subjected to an intensity distribution of the activation light in form of an activation point grating of point-shaped intensity maxima. In the areas with relevant intensity of the activation light, particularly within the intensity maxima of the activation light, the PTOL is activated. Afterwards, i.e. already prior to subjecting the sample to the excitation light, the sample is subjected to a pulse of deactivation light which transfers the PTOL back into its inactive state. The intensity distribution of the deactivation light is formed as a deactivation point grating which is similar to the activation grating. In the deactivation grating, however, intensity minima of the deactivation light are provided at the grating points which are surrounded by shells of higher intensity of the deactivation light. As the deactivation grating is superimposed with the activation grating in such a way that each intensity maximum of the activation light coincides with one intensity minimum of the deactivation light, the PTOL, everywhere outside of the intensity minima of the deactivation light, is transferred back into its inactive state. When the sample is afterwards subjected to an excitation point grating of excitation light which, in the areas of the local maxima of the activation point grating also has local intensity maxima, the PTOL is only excited for the emission of luminescence light in those areas of the intensity minima of the deactivation point grating in which the PTOL is still in its active state. Due to the point grating-shaped illumination with the activation, the deactivation and the excitation light, a parallelized high resolution full image of the structure of the sample is obtained. With regard to the generation of the point grating-shaped intensity distributions, WO 2006/127692 A2 refers to WO 2006/058187 A2.
According to WO 2006/058187 A2, a two- or three-dimensional point grating-shaped intensity distribution in a sample with local intensity maxima at the grating points is generated in that three or four beams of coherent light with different propagation directions are superimposed within the sample such as to form an interference pattern. By adjusting the phases of the individual beams of light it shall be possible to generate local intensity minima at the grating points which are enclosed by areas of higher intensity. Adjusting the phases is achieved by means of a movable reflector, an optical phase modulator or a spatial light modulator (SLM). For generating the point gratings of the activation, the deactivation and the excitation light, pluralities of coherent light beams with different propagation directions are superimposed within the sample so that the desired intensity distribution results from optical interference. According to WO 2006/058187 A2, the point grating-shaped intensity distributions may be used in an STED scanning fluorescence microscope. Here, the sample is subjected to a point grating-shaped intensity distribution of pulsed excitation light with local intensity maxima at the grating points. The excitation light excites a fluorescent marker in the sample for the emission of fluorescence light. Additionally, the sample is subjected to a point grating-shaped intensity distribution of pulsed emission stimulation light having a plurality of local intensity minima. The emission stimulation light, by means of stimulated emission, transfers the fluorescent marker back into its ground state. For generating the intensity distribution of the emission stimulation light with local intensity minima at the grating points which are each surrounded by an area of higher intensity, according to WO 2006/058187 A1, a plurality of similar sub-gratings of the emission stimulation light are generated and superimposed within the sample. Each sub-grating is generated as a three-dimensional point grating with local intensity maxima at the grating points. For each sub-grating three or four beams of light are superimposed whose phases are adjusted with regard to each other in such a way that the respective sub-grating has the desired symmetry and periodicity within the sample. Adapting the phases of the beams of light and thus shifting the sub-gratings with regard to each other, the gratings are then superimposed within the sample in such a way that they all together generate a point grating of local intensity minima. The intensity maxima of the sub-gratings are arranged at an offset in different directions with regard to the intensity minima of the overall grating. The shape of the intensity minima is defined by the relative arrangement of the intensity maxima delimiting the intensity minima. Due to the delimitation of the intensity minima by means of point-shaped or spherical intensity maxima, the intensity minima have no round shape but different extensions in different directions.
A further scanning luminescence light microscope for spatial high resolution examination of samples is disclosed in DE 2006 009 833 B4. Here, means for providing four beams of light of luminescence inhibition light which are coherent in pairs and means for focusing the four beams of light into the pupil of an objective are provided to generate superimposed standing waves in two directions, i.e. two crossing line gratings. The means for providing the four beams of light which are coherent in pairs, for example, include a holographic grating within the beam path. Adjustable retroreflectors are provided for a path length adjustment between the pairs of coherent light beams. An additional light source, like for example for switching or excitation light, illuminates the entire field of view of this known scanning luminescence light microscope.
There is still a need of a scanning luminescence light microscope for spatial high resolution imaging of a structure of a sample comprising a luminescent marker in which the intensity distributions of the luminescence inhibition light and the further light are optimized even over a high number of intensity minima of the luminescence inhibition light.